CC chemokine receptor 5 DNA, new animal models and therapeutic agents for HIV infection

ABSTRACT

The susceptibility of human macrophages to human immunodeficiency virus (HIV) infection depends on cell surface expression of the human CD4 molecule and CC cytokine receptor 5. CCR5 is a member of the 7-transmembrane segment superfamily of G-protein-coupled cell surface molecules. CCRS plays an essential role in the membrane fusion step of infection by some HIV isolates. The establishment of stable, nonhuman cell lines and transgenic mammals having cells that coexpress human CD4 and CCR5 provides valuable tools for the continuing research of HIV infection. In addition, antibodies which bind to CCR5, CCR5 variants, and CCR5-binding agents, capable of blocking membrane fusion between HIV and target cells represent potential anti-HIV therapeutics for macrophage-tropic strains of HIV.

FIELD OF THE INVENTION

The present invention relates to in vitro and in vivo models for thestudy of human immunodeficiency virus (HIV) infection and theeffectiveness of anti-HIV therapeutics. The invention more specificallyrelates to cell surface proteins-that participate in HIV infection andwhich are useful for the development of animal models.

BACKGROUND OF THE INVENTION

An HIV infection cycle begins with the entry of an HIV virus into atarget cell. Entry commences when an HIV envelope glycoprotein (env)binds to a human CD4 molecule in a target cell membrane. This bindingleads to fusion of virus and cell membranes, which in turn facilitatesvirus entry into the host. The HIV-infected host cell eventuallyexpresses env on its surface. This expression allows the infected cellto fuse with uninfected, CD4-positive cells, thereby spreading thevirus.

Recent studies have shown that the HIV fusion process occurs with a widerange of human cell types that either express human CD4 endogenously orthat have been engineered to express human CD4. The fusion process,however, does not occur with nonhuman cell types engineered to expresshuman CD4 even though these nonhuman cells still can bind env. Thedisparity between human and nonhuman cell types exists because membranefusion requires the coexpression of human CD4 and one or more cofactorsspecific to human cell types. Nonhuman cell types that have beenengineered to express human CD4 but not the additionally requiredfactor(s) are incapable of membrane fusion, and therefore arenonperrnissive for HIV infection.

Some individual HIV isolates, designated “macrophage-tropic,”efficiently infect primary macrophages but not immortalized T-celllines. Other isolates, designated “T-cell line-tropic,” have theopposite property and infect immortalized T-cell lines more efficientlythan they infect primary macrophages. Both types of isolates readilyinfect primary T-cells from the body, however. The selective tropism ofthese two types of isolates is thought to be due to their requirementsfor distinct cofactors that are differentially expressed on differentCD4 positive cell types. It should be understood that other HIV strainsare “dual-tropic” and have the ability to infect both macrophages andimmortalized T-cell lines and are believed to be able to use more thanone cofactor.

Recently a cofactor required for fusion of virus and cell membranes hasbeen described Feng et al., Science 272: 872-7 (1996). This factor,called “fusin,” (also known as CXCR4) permits cells that contain humanCD4 to fuse with the surface of an HIV virus. Fusin functionspreferentially for T-cell line-tropic HIV-1 isolates and much less wellfor macrophage-tropic HIV-1 isolates.

The discovery of fusin allows the creation of a successful small animalmodel. Such a model is crucial for studies of HIV infection and of theeffectiveness of anti-HIV therapeutics. But the presence of fusinenables the study of. T-cell line-tropic but not macrophage-tropicisolates. This is an important distinction because macrophage-tropicisolates represent the predominant type of isolates obtained frominfected individuals. Macrophage-tropic isolates also appear to bepreferentially transmitted between individuals. A putative cofactor thatis necessarily expressed with CD4 to allow entry of macrophage-tropicisolates remains unknown.

In recent years, researchers have bred transgenic animals that containcells which express human CD4 and which could be used as models for HIVinfection of macrophages if the macrophage-specific factor were known.See, for example, Dunn et, al., Human immunodeficiency virus type 1infection of human CD4-transgenic rabbits, J. Gen. Vir. 76:1327-1336(1995); Snyder et al., Development and Tissue-Specific Expression ofHuman CD4 in Transgenic Rabbits, Mol. Reprod. & Devel. 40:419-428(1995); Killeen et al., Regulated Expression of Human CD4 Rescues HelperT-Cell Development in Mice Lacking Expression of Endogenous CD4, EMBO J.12:1547-1553 (1993); Forte et al., Human CD4 Produced in Lymphoid Cellsof Transgenic Mice Binds HIV gp120 and Modifies the Subsets of MouseT-Cell Populations, Immunogenetics 38:455-459 (1993).

A goal of research in this field is to find a putative factor for themacrophage-tropic isolates that could be co-expressed with CD4 in asmall animal. Such co-expression would provide an animal model todevelop efficacious therapies to combat infection by macrophage-tropicHIV isolates. The discovery of other essential cofactors would providenew targets for development of anti-HIV therapies.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a new CC chemokinereceptor protein associated with HIV infection (formerly referred to as“CC CKR5”, now more commonly known as “CCR5”). The invention providesisolated polynucleotides and polypeptides encoded by CCR5polynucleotides, as well as antibodies directed against regions of CCR5and peptide fragments of CCR5 which block HIV interaction with the CCCKCR5 receptor.

It is an object of the present invention to provide therapeutic andpreventative medicinal agents effective against HIV infection andeffective in regulating monocyte 10 accumulation and activation. Inaccomplishing these and other objects, there has been provided, inaccordance with one aspect of the present invention a stable, nonhumancell line, the cells of which contain DNA encoding CCR5. In accordancewith another aspect of the invention a transgenic non-human mammal isprovided comprised of cells that coexpress human CD4 and CCR5.

In another aspect of the invention, the invention provides an antibodywhich binds to CCR5 and which blocks membrane fusion between HIV and atarget cell. In accordance with another aspect of the invention, thereis provided a cell that expresses a CCR5 gene, wherein the CCR5 gene isnot stably integrated into the genome of said cell.

In accordance with yet another aspect of the invention an isolated andpurified peptide fragment of CCR5 is provided that blocks membranefusion between HIV and a target cell.

In yet another aspect, the invention provides a method for identifying acompound which blocks membrane fusion between HIV and a CCR5 target cellor between an HIV-infected cell and a CCR5 positive uninfected cell. Themethod includes the steps of: a) incubating components comprising thecompound and a CDU and CCR5 positive cell under conditions sufficient toallow the components to interact; b) contacting the components of stepa) with HIV or an HIV-infected cell; and c) measuring the ability of thecompound to block membrane fusion between HIV and the CCR5 positive cellor between an HIV-infected cell and a CCR5 positive uninfected cell.

In accordance with yet another aspect of the invention a method ofinhibiting CCR5 expression in a cell is provided, comprising introducinginto the cell at least one antisense polynucleotide that causes theinhibition of CCR5 in the cell.

In accordance with yet another aspect of the invention is provided aCCR5-binding agent, wherein said agent blocks binding of a chemokine andHIV to CCR5.

The antibodies and blocking agents of the invention are also useful forproviding methods for modulating an immune response in which macrophagesare involved. For example, administration of CCR5 agonists orantagonists would be useful for modulating the immune response.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an alignment of amino acid sequences deduced from cDNAsfor CC CKR1, CC CKR2B, and for CCR5. Arabic numbers enumerate a CCR5amino acid sequence (SEQ ID NO:4) and a variant with residue changedfrom alanine to leucine (SEQ ID NO: 2) that has been deduced from a CCR5DNA sequence (SEQ ID NO:3 and SEQ ID NO: 1, respectively) and areleft-justified. Putative membrane-spanning segments I-VII are noted.Vertical bars show identities between adjacent residues and open boxesshow predicted sites for N-linked glycosylation. Dashes and gaps havebeen inserted to optimize the alignments. Extracellular portions of theCCR5 polypeptide are located between transmembrane domains 2 and 3,transmembrane domains 4 and 5, transmembrane domains 6 and 7, and in theamino terminal segment before transmembrane domain 1.

FIG. 1B shows the nucleotide and deduced amino acid sequence (SEQ IDNO:1 and 2, respectively) for a CCR5 variant where nucleotides 293-296of the wild-type DNA is changed from CTTG to TGCT resulting in a changeat amino acid residue 127, from Alanine to Leucine.

FIG. 1C shows the nucleotide and deduced amino acid sequence (SEQ IDNO:3 and 4, respectively) for CCR5.

FIG. 2 shows CCR5 peptides which inhibit fusion between cells expressingthe HIV-1 Env from the macrophage-tropic Ba-L isolate and murine cellsco-expressing CD4 and CCR5. Peptides were preincubated withHIV-Env-expressing cells for 1 hour at a concentration from 0-50 μg/mlbefore mixing with cells which express CD4 and CCR5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention originated from studies on receptor proteins ofchemokines. The inventors cloned, sequenced, and functionally expresseda human cDNA encoding a novel macrophage-selective CC chemokine receptorthat has been designated CCR5.

During their investigation, the inventors discovered that CCR5 is anecessary cofactor for infection by macrophage-tropic HIV isolates. Moreparticularly, the inventors found that when they transgenicallyexpressed human CCR5 in non-human cells which also transgenicallyexpress human CD4, the altered cells could fuse with cells that expressthe env envelope protein from macrophage-tropic strains of HIV. Itshould be understood that other HIV strains are “dual-tropic” and havethe ability to infect both macrophages and immortalized T-cell lines andare believed to be able to use more than one cofactor. Furthermore, theinventors reasoned that antibodies against CCR5 can inhibit the fusionof cells that contain CD4 and CCR5, upon contact with cells that expressthe env protein from macrophage-tropic strains of HIV. Antibodies whichbind CCR5 can inhibit infection of cells that contain CCR5 and CD4 bymacrophage-tropic strains of HIV. The insights of the present inventionenable the development of new tools to study HIV infection ofmracrophages and the discovery of new HIV treatment methodologies basedon chemokine receptor biochemistry.

Chemokine receptors are thought to have seven transmembrane-domains, arecoupled to G-protein and participate in cellular responses tochemokines. Receptor CCR5 that has been cloned by the inventors is thefifth human CC chemokine receptor identified to date. The five receptorsbind overlapping but distinct subsets of CC chemokines. Of the five,only CC chemokine receptor 5 (“CCR5”) displays a CC chemokinespecificity profile that matches the profile for suppression of HIV-1infection. Cocchi et al., Science 270, 1811 (1995). RANTES, MIP-1α andMIP-1β are potent agonists of CCR5, but MCP-1 and MCP-3 are not, assummarized by Combadiere et al. in J. Biol. Chem. 270: 16491-4 (1995),J. Biol. Chem. 270: 30235 (1995), and Molec. Biol. Cell. 6: 224a (1995)and by Samson et al. in Biochemistry 35: 3362 (1996) the disclosures ofwhich are incorporated herein in their entireties.

Isolation of cDNA Encoding CCR5

The gene for the chemokine receptor of the present invention can becloned from a human cDNA library. Methods used to clone novel chemokinereceptor-like cDNAs from a λgt11 cDNA library made from peripheral bloodmononuclear cells of a patient with eosinophilic leukemia have beendescribed by Combadiere et al., DNA Cell Biol. 14: 673-80 (1995), whichis herein incorporated in its entirety by reference. A cDNA encodingCCR5 also can be isolated by the procedure described by U.S. provisionalpatent application 60/010,854 filed on Jan. 30, 1996, which is hereinincorporated by reference.

The above-described methods can be used to identify DNA sequences thatcode for one or more CCR5 polypeptide sequences. A nucleotide sequencedetermined by the inventors, herein described as SEQ ID NO:3 of thepresent invention, has been deposited with the Genbank/EMBL datalibraries under accession number U57840. But many other relatedsequences that code for CCR5 and altered forms of CCR5 are contemplatedin context of the various embodiments enumerated herein (e.g., SEQ IDNO:1).

In preferred embodiments fusion between env-expressing effector cellsand CD4-expressing and CCR5-expressing target cells, prepared byinfection with vaccinia virus, induces activation of Escherichia colilacZ, causing β-galactosidase production in fused cells as described byNussbaum et al., J. Virol. 68: 5411 (1994), which is incorporated in itsentirety by reference. The specificity of cell fusion as measured withthis assay is equivalent to the specificity of infection by HIV-1virions.

The invention provides an isolated polynucleotide sequence encoding apolypeptide having an amino acid sequence as set forth in SEQ ID NO:4.The term “isolated” as used herein includes polynucleotidessubstantially free of other nucleic acids, proteins, lipids,carbohydrates or other materials with which it is naturally associated.Polynucleotide sequences of the invention include DNA, cDNA and RNAsequences which encode CCR5. It is understood that all polynucleotidesencoding all or a portion of CCR5 are also included herein, as long asthey encode a polypeptide with CCR5 activity (e.g., act as a cofactorfor HIV infection). Such polynucleotides include naturally occurring,synthetic, and intentionally manipulated polynucleotides. For example,portions of the mRNA sequence may be altered due to alternate RNAsplicing patterns or the use of alternate promoters for RNAtranscription. As another example, CCR5 polynucleotide may be subjectedto site-directed mutagenesis. The polynucleotide sequence for CCR5 alsoincludes antisense sequences. The poly-nucleotides of the inventioninclude sequences that are degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of CCR5polypeptide encoded by the nucleotide sequence is functionallyunchanged. Also included are nucleotide sequences which encode CCR5polypeptide, such as SEQ ID NO:1. In addition, the invention alsoincludes a polynucleotide encoding a polypeptide having the biologicalactivity of an amino acid sequence of SEQ ID NO:4 and having at leastone epitope for an antibody immunoreactive with CCR5 polypeptide. Assaysprovided herein which show association between HIV infection andexpression of CCR5 can be used to detect CCR5 activity.

The polynucleotide encoding CCR5 includes the nucleotide sequence inFIG. 1 (SEQ ID NO: 1 and 3), as well as nucleic acid sequencescomplementary to that sequence. A complementary sequence may include anantisense nucleotide. When the sequence is RNA, the deoxyribonucleotidesA, G, C, and T of FIG. 1 are replaced by ribonucleotides A, G, C, and U,respectively. Also included in the invention are fragments (portions) ofthe above-described nucleic acid sequences that are at least 15 bases inlength, which is sufficient to permit the fragment to selectivelyhybridize to DNA that encodes the protein of FIG. 1 (e.g., SEQ ID NO:4). “Selective hybridization” as used herein refers to hybridizationunder moderately stringent or highly stringent physiological conditions(See, for example, the techniques described in Maniatis et al., 1989Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., incorporated herein by reference), which distinguishes relatedfrom unrelated nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

Specifically-disclosed herein is a cDNA sequence for CCR5. SEQ ID NO:3represents the wild-type sequence and SEQ ID NO:1 represents a cDNAwhich encodes CCR5 having a conservative substitution of Leucine forAlanine at amino acid residue 127. The result of this conservativevariation should not affect biological activity of CCR5 polypeptide orpeptides containing the variation (see Example 5).

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization or computer-basedtechniques which are well known in the art. These include, but are notlimited to: 1) hybridization of genomic or cDNA libraries with probes todetect homologous nucleotide sequences; 2) antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features; 3) polymerase chain reaction (PCR) on genomic DNAor cDNA using primers capable of annealing to the DNA sequence ofinterest; 4) computer searches of sequence databases for similarsequences; and 5) differential screening of a subtracted DNA library.

Preferably the CCR5 polynucleotide of the invention is derived from amammalian organism. Screening procedures which rely on nucleic acidhybridization make it possible to isolate any gene sequence from anyorganism, provided the appropriate probe is available. Oligonucleotideprobes, which correspond to a part of the sequence encoding the proteinin question, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction when the sequence isdegenerate. This includes a heterogeneous mixture of denatureddouble-stranded DNA. For such screening, hybridization is preferablyperformed on either single-stranded DNA or denatured double-strandedDNA. Hybridization is particularly useful in the detection of cDNAclones derived from sources where an extremely low amount of mRNAsequences relating to the polypeptide of interest are present. In otherwords, by using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucl. Acid Res.,9:879, 1981). Alternatively, a subtractive library, as illustratedherein is useful for elimination of non-specific cDNA clones.

When the entire sequence of amino acid residues of the desiredpolypeptide is not known, the direct synthesis of DNA sequences is notpossible and the method of choice is the synthesis of cDNA sequences.Among the standard procedures for isolating cDNA sequences of interestis the formation of plasmid- or phage-carrying cDNA libraries which arederived from reverse transcription of mRNA which is abundant in donorcells that have a high level of genetic expression. When used incombination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for CCR5 peptides having at least one epitope, usingantibodies specific for CCR5. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of CCR5 cDNA.

Alterations in CCR5 nucleic acid include intragenic mutations (e.g.,point mutation, nonsense (stop), missense, splice site and frameshift)and heterozygous or homozygous deletions. Detection of such alterationscan be done by standard methods known to those of skill in the artincluding sequence analysis, Southern blot analysis, PCR based analyses(e.g., multiplex PCR, sequence tagged sites (STSs)) and in situhybridization. Such proteins can be analyzed by standard SDS-PAGE and/orimmunoprecipitation analysis and/or Western blot analysis, for example.

DNA sequences encoding CCR5 can be expressed in vitro by DNA transferinto a suitable host cell. “Host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the CCR5 polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theCCR5 genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

Polynucleotide sequences encoding CCR5 can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. However, since mature CCR5 is glycosylated, thechoice of host cells depends on whether or not the glycosylated ornon-glycosylated form of CCR5 is desired. Methods of expressing DNAsequences having eukaryotic or viral sequences in prokaryotes are wellknown in the art. Biologically functional viral and plasmid DNA vectorscapable of expression and replication in a host are known in the art.Such vectors are used to incorporate DNA sequences of the invention.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the CCR5 coding sequence andappropriate transcriptional/-translational control signals. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo recombination/genetic techniques. (See, forexample, the techniques described in Maniatis et al., 1989 MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.)

A variety of host-expression vector systems may be utilized to expressthe CCR5 coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the CCR5 coding sequence; yeast transformed with recombinantyeast expression vectors containing the CCR5 coding sequence; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the CCR5 coding sequence; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the CCR5 coding sequence; or animal cell systems infectedwith recombinant virus expression vectors (e.g., retroviruses,adenovirus, vaccinia virus) containing the CCR5 coding sequence, ortransformed animal cell systems engineered for stable expression. SinceCCR5 has not been confirmed to contain carbohydrates,both bacterialexpression systems as well as those that provide for translational andpost-translational modifications may be used; e.g., mammalian, insect,yeast or plant expression systems.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see e.g., Bitteret al., 1987, Methods in Enzymology 153:516-544). For example, whencloning in bacterial systems, inducible promoters such as pL ofbacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of the insertedCCR5 coding sequence.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish Assoc. & WileyInterscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol.II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of theYeast Saccharomyces, 1982, Eds. Strathem et al., Cold Spring HarborPress, Vols. I and II. A constitutive yeast promoter such as ADH or LEU2or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch.3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed. DMGlover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may beused which promote integration of foreign DNA sequences into the yeastchromosome.

Eukaryotic systems, and preferably mammalian expression systems, allowfor proper post-translational modifications of expressed mammalianproteins to occur. Eukaryotic cells which possess the cellular machineryfor proper processing of the primary transcript, glycosylation,phosphorylation, and advantageously, plasma membrane insertion of thegene product may be used as host cells for the expression of CCR5.

Mammalian cell systems which utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the CCR5 coding sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. Alternatively, thevaccinia virus 7.5K promoter may be used. (e.g., see, Mackett et al.,1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al., 1984, J.Virol. 49: 857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA79: 4927-4931). Of particular interest are vectors based on bovinepapilloma virus which have the ability to replicate as extrachromosomalelements (Sarver, et al., 1981, Mol. Cell. Biol. 1: 486). Shortly afterentry of this DNA into mouse cells, the plasmid replicates to about 100to 200 copies per cell. Transcription of the inserted cDNA does notrequire integration of the plasmid into the host's chromosome, therebyyielding a high level of expression. These vectors can be used forstable expression by including a selectable marker in the plasmid, suchas, for example, the neo gene. Alternatively, the retroviral genome canbe modified for use as a vector capable of introducing and directing theexpression of the CCR5 gene in host cells (Cone & Mulligan, 1984, Proc.Natl. Acad. Sci. USA 81:6349-6353). High level expression may also beachieved using inducible promoters, including, but not limited to, themetallothionine IIA promoter and heat shock promoters.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withthe CCR5 cDNA controlled by appropriate expression control elements(e.g., promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. The selectablemarker in the recombinant plasmid confers resistance to the selectionand allows cells to stably integrate the plasmid into their chromosomesand grow to form foci which in turn can be cloned and expanded into celllines. For example, following the introduction of foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. A number of selectionsystems may be used, including but not limited to the herpes simplexvirus thymidine kinase (Wigler,et al., 1977, Cell 11: 223),hypoxanthine-guanine phospho-ribosyltransferase (Szybalska & Szybalski,1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) genes canbe employed in tk-, hgprt- or aprt- cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072; neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad Sci. USA 85: 8047); and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987,In: Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.).

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the CCR5 of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Cell Lines

In one embodiment, the present invention relates to stable recombinantcell lines, the cells of which express CCR5 polypeptide or coexpresshuman CD4 and CCR5 and contain DNA that encodes CCR5. Suitable celltypes include but are not limited to cells of the following types: NIH3T3 (Murine), Mv 1 lu (Mink), BS-C-1 (African Green Monkey) and humanembryonic kidney (HEK) 293 cells. Such cells are described, for example,in the Cell Line Catalog of the American Type Culture Collection (ATCC).These cells can be stably transformed by a method known to the skilledartisan. See, for example, Ausubel et al., Introduction of DNA IntoMammalian Cells, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, sections9.5.1-9.5.6 (John Wiley & Sons, Inc. 1995). “Stable” transformation inthe context of the invention means that the cells are immortal to theextent of having gone through at least 50 divisions.

CCR5 can be expressed using inducible or constituitive regulatoryelements for such expression. Commonly used constituitive or induciblepromoters, for example, are known in the art. The desired proteinencoding sequence and an operably linked promoter may be introduced intoa recipient cell either as a non-replicating DNA (or RNA) molecule,which may either be a linear molecule or, more preferably, a closedcovalent circular molecule. Since such molecules are incapable ofautonomous replication, the expression of the desired molecule may occurthrough the transient expression of the introduced sequence.Alternatively, permanent expression may occur through the integration ofthe introduced sequence into the host chromosome. Therefore the cellscan be transformed stably or transiently.

An example of a vector that may be employed is one which is capable ofintegrating the desired gene sequences into the host cell chromosome.Cells which have stably integrated the introduced DNA into theirchromosomes can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may complement an auxotrophy in the host (such asleu2, or ura3, which are common yeast auxotrophic markers), biocideresistance, e.g., antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a. particular plasmidor viral vector include: the ease with which recipient cells thatcontain the vector may be recognized and selected from those recipientcells which do not contain the vector; the number of copies of thevector which are desired in a particular host; and whether it isdesirable to be able to “shuttle” the vector between host cells ofdifferent species.

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilize DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors include vaccinia virus expressionvectors. A third class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers (e.g., an exogenous gene) whichallow selection of host cells which contain the expression vector. Themarker may provide for prototropy to an auxotrophic host, biocideresistance, e.g., antibiotics, or heavy metals, such as copper or thelike. The selectable marker gene can either be directly linked to theDNA sequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals. The cDNAexpression vectors incorporating such elements include those describedby Okayama, H., Mol. Cell. Biol., 3:280 (1983), and others.

Once the vector or DNA sequence containing the construct has beenprepared for expression, the DNA construct may be introduced(transformed) into an appropriate host. Various techniques may beemployed, such as protbplast fusion, calcium phosphate precipitation,electroporation or other conventional techniques.

Transgenic Animals

In another embodiment, the present invention relates to transgenicanimals having cells that coexpress human CD4 and CCR5. Such transgenicanimals represent a model system for the study of HIV infection and thedevelopment of more effective anti-HIV therapeutics.

The term “animal” here denotes all mammalian species except human. Italso includes an individual animal in all stages of development,including embryonic and fetal stages. Farm animals (pigs, goats, sheep,cows, horses, rabbits and the like), rodents (such as mice), anddomestic pets (for example, cats and dogs) are included within the scopeof the present invention.

A “transgenic” animal is any animal containing cells that bear geneticinformation received, directly or indirectly, by deliberate geneticmanipulation at the subcellular level, such as by microinjection orinfection with recombinant virus. “Transgenic” in the present contextdoes not encompass classical crossbreeding or in vitro fertilization,but rather denotes animals in which one or more cells receive arecombinant DNA molecule. Although it is highly preferred that thismolecule be integrated within the animal's chromosomes, the presentinvention also contemplates the use of extrachromosomally replicatingDNA sequences, such as might be engineered into yeast artificialchromosomes.

The term “trarsgenic animal” also includes a “germ cell line” transgenicanimal. A germ cell line transgenic animal is a-transgenic animal inwhich the genetic information has been taken up and incorporated into agerm line cell, therefore conferring the ability to transfer theinformation to offspring. If such offspring in fact possess some or allof that information, then they, too, are transgenic animals.

It is highly preferred that the transgenic animals of the presentinvention be produced by introducing into single cell embryos DNAencoding CCR5 and DNA encoding human CD4, in a manner such that thesepolynucleotides are stably integrated into the DNA of germ line cells ofthe mature animal and inherited in normal mendelian fashion. Advances intechnologies for embryo micromanipulation now permit introduction ofheterologous DNA into fertilized mammalian ova. For instance, totipotentor pluripotent stem cells can be transformed by microinjection, calciumphosphate mediated precipitation, liposome fusion, retroviral infectionor other means, the transformed cells are then introduced into theembryo, and the embryo then develops into a transgenic animal. In apreferred method, developing embryos are infected with a retroviruscontaining the desired DNA, and transgenic animals produced from theinfected embryo.

In a most preferred method the appropriate DNAs are coinjected into-thepronucleus or cytoplasm of embryos, preferably at the single cell stage,and the embryos allowed to develop into mature transgenic animals. Thesetechniques are well known. For instance, reviews of standard laboratoryprocedures for microinjection of heterologous DNAs into mammalian(mouse, pig, rabbit, sheep, goat, cow) fertilized ova include: Hogan etal., MANIPULATlNG THE MOUSE EMBRYO (Cold Spring Harbor Press 1986);Krimpenfort et al., Bio/Technology 9:86 (1991); Palmiter et al., Cell41:343 (1985); Kraemer et al., GENETIC MANIPULATION OF THE EARLYMAMMALIAN EMBRYO (Cold Spring Harbor Laboratory Press 1985); Hammer etal., Nature, 315:680 (1985); Purcel et al., Science, 244:1281 (1986);Wagner et al., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat.No. 5,175,384, the respective contents of which are incorporated byreference.

The cDNA that encodes CCR5 can be fused in proper reading frame underthe transcriptional and translational control of a vector to produce agenetic construct that is then amplified, for example, by preparation ina bacterial vector, according to conventional methods. See, for example,the standard work: Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL (Cold Spring Harbor Press 1989), the contents of which areincorporated by reference. The amplified construct is thereafter excisedfrom the vector and purified for use in producing transgenic animals.

Production of transgenic animals containing the gene for human CD4 havebeen described. See Snyder et al., supra; Dunn et al., supra, thecontents of which are incorporated by reference.

The term “transgenic” as used herein additionally includes any organismwhose genome has been altered by in vitro manipulation of the earlyembryo or fertilized egg or by any transgenic technology to induce aspecific gene knockout. The term “gene knockout” as used herein, refersto the targeted disruption of a gene in vivo with complete loss offunction that has been achieved by any transgenic technology familiar tothose in the art. In one embodiment, transgenic animals having geneknockouts are those in which the target gene has been renderednonfunctional by an insertion targeted to the gene to be renderednon-functional by homologous recombination. As used herein, the term“transgenic” includes any transgenic technology familiar to those in theart which can produce an organism carrying an introduced transgene orone in which an endogenous gene has been rendered non-functional or“knocked out.”

The transgene to be used in the practice of the subject invention is aDNA sequence comprising a modified CCR5 coding sequence. In a preferredembodiment, the CCR5 gene is disrupted by homologous targeting inembryonic stem cells. For example, the entire mature C-terminal regionof the CCR5 gene may be deleted as described in the examples below.Optionally, the CCR5 disruption or deletion may be accompanied byinsertion of or replacement with other DNA sequences, such as anon-functional CCR5 sequence. In other embodiments, the transgenecomprises DNA antisense to the coding sequence for CCR5. In anotherembodiment, the transgene comprises DNA encoding an antibody or receptorpeptide sequence which is able to bind to CCR5. Where appropriate, DNAsequences that encode proteins having CCR5 activity but differ innucleic acid sequence due to the degeneracy of the genetic code may alsobe used herein, as may truncated forms, allelic variants andinterspecies homologues.

Antibodies Which Bind to CCR5 Inhibit Fusion

In another embodiment, the present invention relates to antibodies thatbind CCR5 that block env-mediated membrane fusion (i) associated withHIV entry into a human CD4-positive target cell or (ii) between anHIV-infected cell and an uninfected human CD4-positive target cell. Theinvention also includes antibodies that bind to CCR5 and inhibitchemokine binding. For example, such antibodies may be useful forameliorating immune response disorders associated with macrophages.Antibodies of the invention may also inhibit gp120 binding to CCR5. Suchantibodies could represent research and diagnostic tools in the study ofHIV infection and the development of more effective anti-HIVtherapeutics. In addition, pharmaceutical compositions comprisingantibodies against CCR5 may represent effective anti-HIV therapeutics.

An antibody suitable for blocking env-mediated membrane fusion,inhibiting chemokine binding, or blocking gp120 binding to CCR5, isspecific for at least one portion of an extracellular region of the CCR5polypeptide, as shown in FIG. 1 (SEQ ID NO:2 and 4). For example, one ofskill in the art can use the peptides in SEQ ID NO:5-7 or otherextracellular amino acids of CCR5 to generate appropriate antibodies ofthe invention. Alternatively, one of skill in the art can use wholecells expressing CCR5 as an immunogen for generation of anti-CCR5antibodies which either block env-mediated membrane fusion, inhibitchemokine binding or block gp120 binding to CCR5. Anti-CCR5 antibodiesof the invention may have any or all of these functions.

A target cell includes but is not limited to a cell of the followingtypes: Mv 1 lu, NIH 3T3, BS-C-1, HEK293 cells and primary human T-cellsand macrophages. Antibodies of the invention include polyclonalantibodies, monoclonal antibodies, and fragments of polyclonal andmonoclonal antibodies.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., Production of PolyclonalAntisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (HumanaPress 1992); Coligan et al., Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY,section 2.4.1 (1992), which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORYMANUAL, page 726 (Cold Spring Harbor Pub. 1988), which are herebyincorporated by reference. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verify the presence of antibody production by removing a serum sample,removing the spleen to obtain B lymphocytes, fusing the B lymphocyteswith myeloma cells to produce hybridomas, cloning the hybridomas,selecting positive clones that produce antibodies to the antigen, andisolating the antibodies from the hybridoma cultures. Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al.,Purification of Immunoglobulin G (IgG), in METHODS IN MOLECULAR BIOLOGY,VOL. 10, pages 79-104 (Humana Press 1992).

Methods of in vitro and in vivo multiplication of monoclonal antibodiesis well-known to those skilled in the art. Multiplication in vitro maybe carried out in suitable culture media such as Dulbecco's ModifiedEagle Medium or RPMI 1640 medium, optionally replenished by a mammalianserum such as fetal calf serum or trace elements and growth-sustainingsupplements such as normal mouse peritoneal exudate cells, spleen cells,bone marrow macrophages. Production in vitro provides relatively pureantibody preparations and allows scale-up to yield large amounts of thedesired antibodies. Large scale hybridoma cultivation can be carried outby homogenous suspension culture in an airlift reactor, in a continuousstirrer reactor, or in immobilized or entrapped cell culture.Multiplication in vivo may be carried out by injecting cell clones intomammals histocompatible with the parent cells, e.g., osyngeneic mice, tocause growth of antibody-producing tumors. Optionally, the animals areprimed with a hydrocarbon, especially oils such as pristane(tetramethylpentadecane) prior to injection. After one to three weeks,the desired monoclonal antibody is recovered from the body fluid of theanimal.

Therapeutic applications for antibodies disclosed herein are also partof the present invention. For example, antibodies of the presentinvention may also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in Goldenberg et al., International PatentPublication WO 91/11465 (1991) and Losman et al., Int. J. Cancer 46:310(1990), which are hereby incorporated by reference.

Alternatively, a therapeutically useful anti-CCR5 antibody may bederived from a “humanized” monoclonal antibody. Humanized monoclonalantibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in itsentirety by reference. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321: 522(1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al.,Science 239: 1534 (1988); Carter et at., Proc. Nat'l Acad. Sci USA 89:4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer etal., J. Immunol. 150: 2844 (1993), which are hereby incorporated byreference.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433(1994), which are hereby incorporated by reference. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856(1994); and Taylor et al., Int. Immunol. 6:579 (1994), which are herebyincorporated by reference.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein. These patents are herebyincorporated in their entireties by reference. See also Nisonhoff etal., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119(1959); Edelman et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422(Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent, as described in Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,supra. Preferably, the Fv fragments comprise V_(H) and V_(L) chainsconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and _(L)V domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are described, for example, by Whitlow et al., METHODS: A COMPANIONTO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird et al, Science242:423-426 (1988); Ladner et al., U.S. Pat. No. 4,946,778; Pack et al.,Bio/Technology 11: 1271-77 (1993); and Sandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for-example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

Antibodies that bind to CXCR4 chemokine receptor, another HIV fusioncofactor receptor, have been shown to block fusion of HIV strains thatuse CXCR4 receptor for infection (Feng, et al., Science 272:872, 1996;Endres, et al., Cell 87:745, 1996).

Variants of CCR5

The term “CCR5 variant” as used herein means a molecule that simulatesat least part of the structure of CCR5 and interferes with the fusion ofcells that express env with cells that express CD4 and CCR5. The envprotein of certain HIV isolates may participate in HIV infectivity bybinding to CCR5 at the surface of certain cells. While not wishing to bebound by a particular theory of the invention, the inventors believethat CCR5 variants may interfere in HIV infectivity by competing withthe binding of CCR5 to env. CCR5 variants may also be useful inpreventing chemokine binding, thereby ameliorating symptoms ofmacrophage associated immune disorders.

In one embodiment, the present invention relates to peptides and peptidederivatives that have fewer amino acid residues than CCR5 and that blockmembrane fusion between HIV and a target cell. Such peptides and peptidederivatives could represent research and diagnostic tools in the studyof HIV infection and the development of more effective anti-HIVtherapeutics. The preferred peptide fragments of CCR5 according to theinvention include those which correspond to the regions of CCR5 that areexposed on the cell surface (e.g., SEQ ID NO:5, 6 or 7).

The invention relates not only to peptides and peptide derivatives ofnaturally-occurring CCR5, but also to CCR5 mutants and chemicallysynthesized derivatives of CCR5 that block membrane fusion between HIVand a target cell. For example, changes in the amino acid sequence ofCCR5 are contemplated in the present invention. CCR5 can be altered bychanging the DNA encoding the protein (e.g., SEQ ID NO:1&2). Preferably,only conservative amino acid alterations are undertaken, using aminoacids that have the same or similar properties. Illustrative amino acidsubstitutions include the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparggine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine,glutamine, or glutamate; methionine to leucine or isoleucine;phenylalanine to tyrosine, leucine or methionine; serine to threonine;threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan orphenylalanine; valine to isoleucine or leucine.

Variants useful for the present invention comprise analogs, homologs,muteins and mimetics of CCR5 that retain the ability to block membranefusion. Peptides of the CCR5 refer to portions of the amino acidsequence of CCR5 that also retain this ability. The variants can begenerated directly from CCR5 itself by chemical modification, byproteolytic enzyme digestion, or by combinations thereof. Additionally,genetic engineering techniques, as well as methods of synthesizingpolypeptides directly from amino acid residues, can be employed.

Peptides of the invention include the following which correspond toextracellular loops of CCR5 (amino acid designations are according tothe single letter code): extracellular loop-1 (el-1): AILAAQWDFGNTMC(SEQ ID NO:4) extracellular loop-2 (el-2): RSQKEGLHYTCSSHFPYSQYQFWK (SEQID NO:5) extracellular loop-3 (el-3): QEFFGLNNCSSSNRLD (SEQ ID NO:6)FIG. 2 shows the ability of SEQ ID NO: 4, 5 and 6 to inhibit fusionbetween cells expressing the HIV-1 env (from the macrophage tropic Ba-Lisolate) and murine cells co-expressing CD4 and CCR5.

Peptides of the invention can be synthesized by such commonly usedmethods as t-BOC or FMOC protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C terminus of the peptide (See, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods described Merrifield, J. Am. Chem.Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp.27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectros-copy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

Alternatively, peptides can be produced by recombinant methods asdescribed below.

The term “substantially purified” as used herein refers to a molecule,such as a peptide that is substantially free of other proteins, lipids,carbohydrates, nucleic acids, and other biological materials with whichit is naturally associated. For example, a substantially pure molecule,such as a polypeptide, can be at least 60%, by dry weight, themolecule-of interest. One skilled in the art can purify CCR5 peptidesusing standard protein purification methods and the purity of thepolypeptides can be determined using standard methods including, e.g.,polyacrylamide gel electrophoresis (e.g., SDS-PAGE), columnchromatography (e.g., high performance liquid chromatography (HPLC)),and amino-terminal amino acid sequence analysis.

Non-peptide compounds that mimic the binding and function of CCR5(“mimetics”) can be produced by the approach outlined in Saragovi etal., Science 253: 792-95 (1991). Mimetics are molecules which mimicelements of protein secondary structure. See, for example, Johnson etal.,“Peptide Turn Mimetics,” in BIOTECHNOLOGY AND PHARMACY, Pezzuto etal., Eds., (Chapman and Hail, New York 1993). The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions. For the purposes of the presentinvention, appropriate mimetics can be considered to be the equivalentof CCR5 itself.

Longer peptides can be produced by the “native chemical” ligationtechnique which links together peptides (Dawson, et al., Science,266:776, 1994). Variants can be created by recombinant techniquesemploying genomic or cDNA cloning methods. Site-specific andregion-directed mutagenesis techniques can be employed. See CURRENTPROTOCOLS IN MOLECULAR BIOLOGY vol. 1, ch. 8 (Ausubel et al. eds., J.Wiley & Sons 1989 & Supp. 1990-93); PROTEIN ENGINEERING (Oxender & Foxeds., A. Liss, Inc. 1987). In addition, linker-scanning and PCR-mediatedtechniques can be employed for mutagenesis. See PCR TECHNOLOGY (Erliched., Stockton Press 1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vols.1 & 2, supra. Protein sequencing, structure and modeling approaches foruse with any of the above techniques are disclosed in PROTEINENGINEERING, loc. cit., and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,vols. 1 & 2, supra

If the compounds described above are employed, the skilled artisan canroutinely insure that such compounds are amenable for use with thepresent invention in view of the vaccinia cell fusion system describedherein. If a compound blocks env-mediated membrane fusion (i) involvedin HIV entry into a human CD4-positive target cell or (ii) between anHIV-infected cell and an uninfected human CD4-positive target cell, thecompound is suitable according to the invention.

CCR5-Binding and Blocking Agents

In yet another embodiment, the present invention relates to CCR5-bindingagents that block membrane fusion between HIV and a target cell. Suchagents could represent research and diagnostic tools in the study of HIVinfection and the development of more effective anti-HIV therapeutics.In addition, pharmaceutical compositions comprising CCR5-binding agentsmay represent effective anti-HFV therapeutics. In the context of HIVinfection, the phrase “CCR5-binding agent” denotes a naturally occurringligand of CCR5 such as, for example: RANTES, MIP-1α or MIP-1β; asynthetic ligand of CCR5, or appropriate derivatives of the natural orsynthetic ligands. The determination and isolation of ligands is welldescribed in the art. See, e.g., Lemer, Trends NeuroSci. 17:142-146(1994) which is hereby incorporated in its entirety by reference. ACCR5-binding agent that blocks env-mediated membrane fusion (i) involvedin HIV entry into a human CD4-positive target cell or (ii) between anHIV-infected cell and an uninfected human CD4-positive target cell issuitable according to the invention. Further, a CCR5 blocking or bindingagent includes an agent which inhibits gp120 binding to CCR5 orchemokine binding to CCR5.

In yet another embodiment, the present invention relates to CCR5-bindingagents that interfere with binding between CCR5 and a chemokine. Suchbinding agents may interfere by competitive inhibition, bynon-competitive inhibition or by uncompetitive inhibition.

Interference with normal binding between CCR5 and one or more chemokinescan result in a useful pharmacological effect related to inflammationbecause CCR5 binds chemokines that regulate monocyte accumulation andactivation in inflamed tissue sites. Nevertheless, while monocytechemotaxis is the most widely shared and perhaps best described functionfor MIP-1α, MIP-1β and RANTES, apparently each of the CC CKRs that bindone or more of these chemokines connect specifically and differentiallyto additional monocyte functions such as T-cell costimulation.

Monocytes are long-lived cells capable of further differentiation asthey move from the blood to establish residence in the tissues asmacrophages. The functional properties of tissue macrophages differ indifferent organs, and in the same organ depending on the presence ofpriming agents, i.e., agents that can change the behavior of monocytesand make them more sensitive to chemoattractants. CCR5-binding orblocking agents can interfere with the normal functioning of this systemto reduce inflammation and are contemplated by the present invention.Anti-CCR5 antibodies of the invention are also useful in this context.

Screen for CCCKR5 Binding and Blocking Compositions

In another embodiment, the invention provides a method for identifying acomposition which binds to CCR5 or blocks HIV env-mediated membranefusion. The method includes incubating components comprising thecomposition and CCR5 under conditions sufficient to allow the componentsto interact and measuring the binding of the composition to CCR5.Compositions that bind to CCR5 include peptides, peptidomimetics,polypeptides, chemical compounds and biologic agents as described above.In addition to inhibition of cell fusion, one of skill in the art couldscreen for inhibition of gp120 binding or inhibition of CCR5 binding toa chemokine to determine if a compound or composition was a CCR5 bindingor blocking agent.

Incubating includes conditions which allow contact between the testcomposition and CCR5. Contacting includes in solution and in solidphase. The test ligand(s)/composition may optionally be a combinatoriallibrary for screening a plurality of compositions. Compositionsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs)(Landegren, et al., Science, 241:1077, 1988), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren, et al.,Science, 242:229-237, 1988).

To determine if a composition can functionally complex with the receptorprotein, induction of the exogenous gene is monitored by monitoringchanges in the protein levels of the protein encoded for by theexogenous gene, for example. When a composition(s) is found that caninduce transcription of the exogenous gene, it is concluded that thiscomposition(s) can bind to the receptor protein coded for by the nucleicacid encoding the initial sample test composition(s).

Expression of the exogenous gene can be monitored by a functional assayor assay for a protein product, for example. The exogenous gene istherefore a gene which will provide an assayable/measurable expressionproduct in order to allow detection of expression of the exogenous gene.Such exogenous genes include, but are not limited to, reporter genessuch as chloramphenicol acetyltransferase gene, an alkaline phosphatasegene, beta-galactosidase,a luciferase gene, a green fluorescent proteingene, guanine xanthine phosphoribosyltransferase, alkaline phosphatase,and antibiotic resistance genes (e.g., neomycin phosphotrnsferase).

Expression of the exogenous gene is indicative of composition-receptorbinding, thus, the binding or blocking composition can be identified andisolated. The compositions of the present invention can be extracted andpurified from the culture media or a cell by using known proteinpurification techniques commonly employed, such as extraction,precipitation, ion exchange chromatography, affinity chromatography, gelfiltration and the like. Compositions can be isolated by affinitychromatography using the modified receptor protein extracellular domainbound to a column matrix or by heparin chromatography.

Also included in the screening method of the invention is combinatorialchemistry methods for identifying chemical compounds that bind to CCR5.Ligands/compositions that bind to CCR5 can be assayed in standardcell:cell fusion assays, such as the vaccinia assay described herein todetermine whether the composition inhibits or blocks env-mediatedmembrane fusion (i) involved in HIV entry into a human CD4-positivetarget cell or (ii) between an HIV-infected cell and an uninfected humanCD4-positive target cell. Screening methods include inhibition ofchemokine binding to CCR5 (e.g., use radiolabeled chemokine) orinhibition of labeled gp120. For example, a derivative of RANTES wasshown to act as a CCR5 receptor antagonist (RANTES 9-68;Arenzana-Selsdedos et al., Nature 383:400,-1996, incorporated byreference). AOP-RANTES and Met-RANTES were shown to bind with highaffinity yet failed to induce chemotaxis signalling, thereby acting asan antagonist (Simmons et al., Science 276:276, 1997). Thus, thescreening method is also useful for identifying variants, binding orblocking agents, etc., which functionally, if not physically (e.g.,sterically) act as antagonists or agonists, as desired.

Pharmaceutical Compositions

The invention also includes various pharmaceutical compositions thatblock membrane fusion between HIV and a target cell. The pharmaceuticalcompositions according to the invention are prepared by bringing anantibody against CCR5, a peptide or peptide derivative of CCR5, a CCR5mimetic, or a CCR5-binding agent according to the present invention intoa form suitable for administration to a subject using carriers,excipients and additives or auxiliaries. Frequently used carriers orauxiliaries include magnesium carbonate, titanium dioxide, lactose,mannitol and other sugars, talc, milk protein, gelatin, starch,vitamins, cellulose and its derivatives, animal and vegetable oils,polyethylene glycols and solvents, such as sterile water, alcohols,glycerol and polyhydric alcohols. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's The Pharmacological Basis for Therapeutics (7thed.).

In another embodiment, the invention relates to a method of blocking themembrane fusion between HIV and a target cell. This method involvesadministering to a subject a therapeutically effective dose of apharmaceutical composition containing the compounds of the presentinvention and a pharmaceutically acceptable carrier. “Administering” thepharmaceutical composition of the present invention may be accomplishedby any means known to the skilled artisan. By “subject” is meant anymammal, preferably a human.

The pharmaceutical compositions are preferably prepared and administeredin dose units. Solid dose units are tablets, capsules and suppositories.For treatment of a patient, depending on activity of the compound,manner of administration, nature and severity of the disorder, age andbody weight of the patient, different daily doses are necessary. Undercertain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The pharmaceutical compositions according to the invention are ingeneral administered topically, intravenously, orally or parenterally oras implants, but even rectal use is possible in principle. Suitablesolid or liquid pharmaceutical preparation forms are, for example,granules, powders, tablets, coated tablets, (micro)capsules,suppositories, syrups, emulsions, suspensions, creams, aerosols, dropsor injectable solution in ampule form and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, flavorings, sweeteners orsolubilizers are customarily used as described above. The pharmaceuticalcompositions are suitable for use in a variety of drug delivery systems.For a brief review of present methods for drug delivery, see Langer,Science, 249: 1527-1533 (1990), which is incorporated herein byreference.

The pharmaceutical compositions according to the invention may beadministered locally or systemically. By “therapeutically effectivedose” is meant the quantity of a compound according to the inventionnecessary to prevent, to cure or at least partially arrest the symptomsof the disease and its complications. Amounts effective for this usewill, of course, depend on the severity of the disease and the weightand general state of the patient. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof the pharmaceutical composition, and animal models may be used todetermine effective dosages for treatment of particular disorders.Various considerations are described, e.g., in Gilman et al. (eds.)(1990) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS,8th ed., Pergamon Press; and REMINGTON'S PHARMACEUTICAL SCIENCES, 17thed. (1990), Mack Publishing Co., Easton, Pa., each of which is hereinincorporated by reference.

Testing for Newpharmaceutical Compositions

In a preferred embodiment, the invention is a method for screening acompound (“test substance”) for anti-HIV pharmacological activity. Inthis embodiment, the CCR5 and CD4 genes are expressed in one type ofeukaryotic cell and incubated with a second type of eukaryotic cell thatexpresses an HIV envelope protein (“env”). Fusion between at least onecell of each type with the other type is then monitored. The testsubstance is added to the incubation solution before or after mixing ofthe cells and its effect on the fusion rate of cells is determined byany of a number of means. One means to monitor fusion is to include asystem that results in the production of an active P-galactosidase uponcell fusion as described in Nussbaum et al., 1994, supra. If the testmolecule inhibits HIV infectivity then the presence of the molecule willdecrease the cell fusion response. In the case where the test substancebinds a naturally occurring molecule present in the human that isnecessary for HIV infectivity, then addition of the test molecule maydecrease cell fusion.

The cell fusion assay can be used to determine the functional ability ofCCR5 to confer env-mediated fusion competence to a diverse range ofCD4-positive (e.g., either recombinantly produced or naturallyoccurring) cell types: e.g., NIH 3T3 (nurrne); BS-C-1 (African greenmonkey); HEK293 (human); and Mv 1 Lu (mink). In addition, unusual,fusion-incompetent, CD4-positive human cell types can be employed (U-87MG glioblastoma; and SCL1).

Variations of drug screening methods are known to the artisan of averageskill in this field. Consequently, the cell fusion assay can be used ina wide variety of formats to exploit the properties of the CCR5 receptorto screen for drugs that are effective against HIV.

Antisense or Ribozyme Inhibition of CCR5 for HIV Therapy

Antisense technology offers a very specific and potent means ofinhibiting HIV infection of cells that contain CCR5, for example, bydecreasing the amount of CCR5 expression in a cell. Antisensepolynucleotides in context of the present invention includes both shortsequences of DNA known as oligonucleotides of usually 10-50 bases inlength as well as longer sequences of DNA that may exceed the length ofthe CCR5 gene sequence itself. Antisense polynucleotides useful for thepresent invention are complementary to specific regions of acorresponding target mRNA. Hybridization of antisense polynucleotides totheir target transcripts can be highly specific as a result ofcomplementary base pairing. The capability of antisense polynucleotidesto hybridize is affected by such parameters as length, chemicalmodification and secondary structure of the transcript which caninfluence polynucleotide access to the target site. See Stein et al,Cancer Research 48:2659 (1988). An antisense polynucleotide can beintroduced to a cell by introducing a DNA segment that codes for thepolynucleotide into the cell such that the polynucleotide is made insidethe cell. An antisense polynucleotide can also be introduced to a cellby adding the polynucleotide to the environment of the cell such thatthe cell can take up the polynucleotide directly. The latter route ispreferred for the shorter polynucleotides of up to about 20 bases inlength.

In selecting the preferred length for a given polynucleotide, a balancemust be struck to gain the most favorable characteristics. Shorterpolynucleotides such as 10-to 15-mers, while offering higher cellpenetration, have lower gene specificity. In contrast, while longerpolynucleotides of 20-30 bases offer better specificity, they showdecreased uptake kinetics into cells. See Stein et al., PHOSPHOROTHIOATEOLIGODEOXYNUCLEOTIDE ANALOGUES in “Oligodeoxynucleotides—AntisenseInhibitors of Gene Expression” Cohen, ed. McMillan Press, London (1988).Accessibility to mRNA target sequences also is of importance and,therefore, loop-forming regions in targeted mRNAs offer promisingtargets.

In this disclosure the term “polynucleotide” encompasses both oligomericnucleic acid moieties of the type found in nature, such as thedeoxyribonucleotide and ribonucleotide structures of DNA and RNA, andman-made analogues which are capable of binding to nucleic acids foundin nature. The polynucleotides of the present invention can be basedupon ribonucleotide or deoxyribonucleotide monomers linked byphosphodiester bonds, or by analogues linked by methyl phosphonate,phosphorothioate, or other bonds. They may also comprise monomermoieties which have altered base structures or other modifications, butwhich still retain the ability to bind to naturally occurring DNA andRNA structures. Such polynucleotides may be prepared by methodswell-known in the art, for instance using commercially availablemachines and reagents available from Perkin-Elmer/Applied Biosystems(Foster City, Calif.).

Phosphodiester-linked polynucleotides are particularly susceptible tothe action of nucleases in serum or inside cells, and therefore in apreferred embodiment the polynucleotides of the present invention arephosphorothioate or methyl phosphonate-linked analogues, which have beenshown to be nuclease-resistant. Persons of ordinary skill in this artwill be able to select other linkages for use in the invention. Thesemodifications also may be designed to improve the cellular uptake andstability of the polynucleotides.

In another embodiment of the invention, the antisense polynucleotide isan RNA molecule produced by introducing an expression construct into thetarget cell. The RNA molecule thus produced is chosen to have thecapability to hybridize to CCR5 mRNA. Such molecules that have thiscapability can inhibit translation of the CCRS mRNA and thereby inhibitthe ability of HIV to infect cells that contain the RNA molecule.

The polynucleotides which have the capability to hybridize with mRNAtargets can inhibit expression of corresponding gene products bymultiple mechanisms. In “translation arrest,” the interaction ofpolynucleotides with target mRNA blocks the action of the ribosomalcomplex and, hence, prevents translation of the messenger RNA intoprotein. Haeuptle et al., Nucl. Acids. Res. 14:1427 (1986). In the caseof phosphodiester or phosphorothioate DNA polynucleotides, intracellularRNase H can digest the targeted RNA sequence once it has hybridized tothe DNA oligomer. Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988). As a further mechanism of action, in “transcription arrest” itappears that some polynucleotides can form “triplex,” or triple-helicalstructures with double stranded genomic DNA containing the gene ofinterest, thus interfering with transcription by RNA polymerase.Giovannangeli et al., Proc. Natl. Acad Sci. 90:10013(1993); Ebbinghauset al. J. Clin. Invest. 92:2433 (1993).

In one preferred embodiment, CCR5 polynucleotides are synthesizedaccording to standard methodology. Phosphorothioate modified DNApolynucleotides typically are synthesized on automated DNA synthesizersavailable from a variety of manufacturers. These instruments are capableof synthesizing nanomole amounts of polynucleotides as long as 100nucleotides. Shorter polynucleotides synthesized by modern instrumentsare often suitable for use without fer purification. If necessary,polynucleotides may be purified by polyacrylamide gel electrophoresis orreverse phase chromatography. See Sambrook et al., MOLECULAR CLONING: ALaboratory Manual, Vol. 2, Chapter 11, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

Alternatively, a CCR5 polynucleotide in the form of antisense RNA may beintroduced to a cell by its expression within the cell from a standardDNA expression vector. CCR5 DNA antisense sequences can be cloned fromstandard plasmids into expression vectors, which expression vectors havecharacteristics permitting higher levels of, or more efficientexpression of the resident polynucleotides. At a minimum, theseconstructs require a prokaryotic or eukaryotic promoter sequence whichinitiates transcription of the inserted DNA sequences. A preferredexpression vector is one where the expression is inducible to highlevels. This is accomplished by the addition of a regulatory regionwhich provides increased transcription of downsteam sequences in theappropriate host cell. See Sambrook et al., Vol. 3, Chapter 16 (1989).

For example, CCR5 antisense expression vectors can be constructed usingthe polymerase chain reaction (PCR) to amplify appropriate fragmentsfrom single-stranded cDNA of a plasmid such as pRc in which CCR5 cDNAhas been incorporated. Fang et al., J. Biol. Chem. 267 25889-25897(1992). Polynucleotide synthesis and purification techniques aredescribed in Sambrook et al. and Ausubel et al. (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience 1987) (hereafter“Ausubel”), respectively. The PCR procedure is performed via well-knownmethodology. See, for example, Ausubel, and Bangham, “The PolymeraseChain Reaction: Getting Started,” in PROTOCOLS IN HUMAN MOLECULARGENETICS (Humana Press 1991). Moreover, PCR kits can be purchased fromcompanies such as Stratagene Cloning Systems (La Jolla, Calif.) andInvitrogen (San Diego, Calif.).

The products of PCR are subcloned into cloning vectors. In this context,a “cloning vector” is a DNA molecule, such as a plasmid, cosmid orbacteriophage, that can replicate autonomously in a host prokaryoticcell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Suitable cloning vectors aredescribed by Sambrook et al., Ausubel, and Brown (ed.), MOLECULARBIOLOGY LABFAX (Academic Press 1991). Cloning vectors can be obtained,for example, from GIBCO/BRL (Gaithersburg, Md.), Clontech Laboratories,Inc. (Palo Alto, Calif.), Promega Corporation (Madison, Wis.),Stratagene Cloning Systems (La Jolla, Calif.), Invitrogen (San Diego,Calif.), and the American Type Culture Collection (Rockville, Md.).

Preferably, the PCR products are ligated into a “TA” cloning vector.Methods for generating PCR products with a thymidine or adenine overhangare well-known to those of skill in the art. See, for example, Ausubelat pages 15.7.1-15.7.6. Moreover, kits for performing TA cloning can bepurchased from companies such as Invitrogen (San Diego, Calif.).

Cloned antisense fragments are amplified by transforming competentbacterial cells with a cloning vector and growing the bacterial hostcells in the presence of the appropriate antibiotic. See, for example,Sambrook et al., and Ausubel. PCR is then used to screen bacterial hostcells for CCR5 antisense orientation clones. The use of PCR forbacterial host cells is described, for example, by Hofmann et al.,“Sequencing DNA Amplified Directly from, a Bacterial Colony,” in PCRPROTOCOLS: METHODS AND APPLICATIONS, White (ed.), pages 205-210 (HumanaPress 1993), and by Cooper et al., “PCR-Based Full-Length cDNA CloningUtilizing the Universal-Adaptor/Specific DOS Primer-Pair Strategy,” Id.at pages 305-316.

Cloned antisense fragments are cleaved from the cloning vector andinserted into an expression vector. For example, HindIII and XbaI can beused to cleave the antisense fragment from TA cloning vector pCR™-II(Invitrogen;San Diego, Calif.). Suitable expression vectors typicallycontain (1) prokaryotic DNA elements coding for a bacterial origin ofreplication and an antibiotic resistance marker to provide for theamplification and selection of the expression vector in a bacterialhost; (2) DNA elements that control initiation of transcription, such asa promoter; and (3) DNA elements that control the processing oftranscripts, such as a transcription termination/polyadenylationsequence.

For a mammalian host, the transcriptional and translational regulatorysignals preferably are derived from viral sources, such as adenovirus,bovine papilloma virus, simian virus, or the like, in which theregulatory signals are associated with a particular gene which has ahigh level of expression. Suitable transcriptional and translationalregulatory sequences also can be obtained from mammalian genes, such asactin, collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1: 273 (1982)); the TKpromoter of Herpes virus (McKnight, Cell 31: 355 (1982)); the SV40 earlypromoter (Benoist et al., Nature 290: 304 (1981); the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79: 6777 (1982));and the cytomegalovirus promoter (Foecking et al. Gene 45: 101. (1980)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control fusion gene expression ifthe prokaryotic promoter is regulated by a eukaryotic promoter. Zhou etal., Mol. Cell. Biol. 10: 4529 (1990); Kaufman et al., Nucl. Acids Res.19: 4485 (1991).

A vector for introducing at least one antisense polynucleotide into acell by expression from a DNA is the vector pRc/CMV (Invitrogen (SanDiego, Calif.), which provides a high level of constitutivetranscription from mammalian enhancer-promoter sequences. Cloned CCR5antisense vectors are amplified in bacterial host cells, isolated fromthe cells, and analyzed as described above.

Another possible method by which antisense sequences may be exploited isvia gene therapy. Virus-like vectors, usually derived from retroviruses,may prove useful as vehicles for the importation and expression ofantisense constructs in human cells. Generally, such vectors arenon-replicative in vivo, precluding any unintended infection ofnon-target cells. In such cases, helper cell lines are provided whichsupply the missing replicative functions in vitro, thereby permittingamplification and packaging of the antisense vector. A furtherprecaution against accidental infection of non-target cells involves theuse of target cell-specific regulatory sequences. When under the controlof such sequences, antisense constructs would not be expressed in normaltissues.

Two prior studies have explored the feasibility of using antisensepolynucleotides to inhibit the expression of a heparin binding growthfactor. Kouhara et al., Oncogene 9: 455-462 (1994); Morrison, J. Biol.Chem. 266: 728 (1991). Kouhara et al. showed that androgen-dependentgrowth of mouse mammary carcinoma cells (SC-3) is mediated throughinduction of androgen-induced, heparin binding growth factor (AIGF). Anantisense 15-mer corresponding to the translation initiation site ofAIGF was measured for its ability to interfere with androgen-inductionof SC-3 cells. At concentrations of 5 μM, the antisense polynucleotideeffectively inhibited androgen-induced DNA synthesis. Morrison showedthat antisense polynucleotides targeted against basic fibroblast growthfactor can inhibit growth of astrocytes in culture. Thus, the generalfeasibility of targeting an individual gene product in a mammalian cellhas been established.

Antisense polynucleotides according to the present invention are derivedfrom any portion of the open reading frame of the CCR5 cDNA. Preferably,mRNA sequences (i) surrounding the translation initiation site and (ii)forming loop structures are targeted. Based upon the size of the humangenome, statistical studies show that a DNA segment approximately 14-15base pairs long will have a unique sequence in the genome. To ensurespecificity of targeting CCR5 RNA, therefore, it is preferred that theantisense polynucleotides are at least 15 nucleotides in length. Thus,the shortest polynucleotides contemplated by the present inventionencompass nucleotides corresponding to positions 1-14, 1-15, 1-16, 1-17,1-18, 1-19, 2-16, 3-17, etc. of the CCR5 cDNA sequence. Position 1refers to the first nucleotide of the CCR5 coding region.

Not every antisense polynucleotide will provide a sufficient degree ofinhibition or a sufficient level of specificity for the CCR5 target.Thus, it will be necessary to screen polynucleotides to determine whichhave the proper antisense characteristics. A preferred method to assayfor a useful antisense polynucleotide is the inhibition of cell fusionbetween: (1) cells that contain CD4 and CCR5; and (2) cells that containenv.

Administration of an antisense polynucleotide to a subject, either as anaked, synthetic polynucleotide or as part of an expression vector, canbe effected via any common route (oral, nasal, buccal, rectal, vaginal,or topical), or by subcutaneous, intramuscular, intraperitoneal, orintravenous injection. Pharmaceutical compositions of the presentinvention, however, are advantageously administered in the form ofinjectable compositions. A typical composition for such purposecomprises a pharmaceutically acceptable solvent or diluent and othersuitable, physiologic compounds. For instance, the composition maycontain polynucleotide and about 10 mg of human serum albumin permilliliter of a phosphate buffer containing NaCl.

As much as 700 milligrams of antisense polynucleotide has beenadministered intravenously to a patient over a course of 10 days (ie.,0.05 mg/kg/hour) without signs of toxicity. Sterling, “SystemicAntisense Treatment Reported,” Genetic Engineering News 12:. 1, 28(1992).

Other pharmaceutically acceptable excipients include non-aqueous oraqueous solutions and non-toxic compositions including salts,preservatives, buffers and the like. Examples of non-aqueous solutionsare propylene glycol, polyethylene glycol, vegetable oil and injectableorganic esters such as ethyloleate. Aqueous solutions include water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, etc. Intravenous vehicles includefluid and nutrient replenishers. Preservatives include antimicrobial,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to routine skills in the art. A preferredpharmaceutical composition for topical administration is a dermal creamor transdermal patch.

Antisense polynucleotides or their expression vectors may beadministered by injection as an oily suspension. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate or triglycerides.Moreover, antisense polynucleotides or vectors may be combined with alipophilic carrier such as any one of a number of sterols includingcholesterol, cholate and deoxycholic acid. A preferred sterol ischolesterol. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension include, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. Optionally, thesuspension also contains stabilizers.

An alternative formulation for the administration of antisense CCR5polynucleotides involves liposomes. Liposome encapsulation provides analternative formulation for the administration of antisense CCR5polynucleotides and expression vectors. Liposomes are microscopicvesicles that consist of one or more lipid bilayers surrounding aqueouscompartments. See, generally, Bakker-Woudenberg et al., Eur. J. Clin.Microbiol. Infect. Dis. 12 (Suppl. 1): S61 (1993), and Kim, Drugs 46:618 (1993). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s).

See, for example, Machy et al., LIPOSOMES IN CELL BIOLOGY ANDPHARMACOLOGY (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46: 1576 (1989). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents. Scherphofet al., Ann. N. Acad. Sci. 446: 368(1985).

After intravenous administration, conventional liposomes arepreferentially phagocytosed into the reticuloendothelial system.However, the reticuloendothelial system can be circumvented by severalmethods including saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means. Claassen etal., Biochim. Biophys. Acta 802: 428 (1984). In addition, incorporationof glycolipid- or polyethelene glycol-derivatised phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system. Allen et al., Biochim.Biophys. Acta 1068: 133 (1991); Allen et al., Biochim. Biohys. Acta1150: 9 (1993) These Stealth® liposomes have an increased circulationtime and an improved targeting to tumors in animals. Woodle et al.,Proc. Amer. Assoc. Cancer Res. 33: 2672 (1992). Human clinical trialsare in progress, including Phase III clinical trials against Kaposi'ssarcoma. Gregoriadis et al., Drugs 45: 15 (1993).

Antisense polynucleotides and expression vectors can be encapsulatedwithin liposomes using standard techniques. A variety of differentliposome compositions and methods for synthesis are known to those ofskill in the art. See, for example, U.S. Pat. No. 4,844,904, U.S. Pat.No. 5,000,959, U.S. Pat. No. 4,863,740, and U.S. Pat. No. 4,975,282, allof which are hereby incorporated by reference.

Liposomes can be prepared for targeting to particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For instance, antibodies specific to tumorassociated antigens may be incorporated into liposomes, together withantisense polynucleotides or expression vectors, to target the liposomemore effectively to the tumor cells. See, for example, Zelphati et al.,Antisense Research and Development 3: 323-338 (1993), describing the use“immunoliposomes” containing antisense polynucleotides for humantherapy.

In general, the dosage of administered liposome-encapsulated antisensepolynucleotides and vectors will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Dose ranges for particular formulations can bedetermined by using a suitable animal model.

The above approaches can also be used not only with antisense nucleicacid, but also with ribozymes, or triplex agents to block transcriptionor translation of a specific CCRS mRNA, either by masking that mRNA withan antisense nucleic acid or triplex agent, or by cleaving it with aribozyme.

Use of an oligonucleotide to stall transcription is known as the triplexstrategy since the oligomer winds around double-helical DNA, forming athree-strand helix. Therefore, these triplex compounds can be designedto recognize a unique site on a chosen gene (Maher, et al., AntisenseRes. and Dev., 1(3):227, 1991; Helene, C., Anticancer Drug Design,6(6):569, 1991).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that the sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

Homozygous and Heterozygous Mutations in CCR5

It is known that in some cases, a homozygous or heterozygous mutation ina polypeptide or a regulatory region of a gene confers a molecular basisfor a difference in function. Bertina, et al and Greengard, et al.(Bertina, et al., Nature. 369:64, 1994; Greengard, et al., Lancet,343:1361, 1994), first identified the molecular basis for the FVabnormality. The phenotype of APC resistance was shown to be associatedwith heterozygosity or homozygosity for a single point mutation in theFV gene that resulted in the substitution of arginine at amino acidresidue 506 with glutamine (FV R506Q). This R506Q mutation prevents APCfrom cleaving a peptide bond at Arg-506 in FV that is required toinactivate factor Va (Bertina, supra; Sun, et al., Blood, 83:3120,1994).

Similarly, the present invention envisions diagnostic and prognostic,and in addition, therapeutic approaches to treatment of HIV-associatedsyndromes based on homozygosity or heterozygosity of CCR5 mutants. Forexample, while not wanting to be bound by a particular theory, it isbelieved that a subject having a homozygous mutant of CCR5 may be HIVresistant or exhibit a slower rate of disease progression. Along thesame lines, a subject having a heterozygous mutation in CCR5 may exhibita slower rate of disease progression than a patient having a wild typeCCR5. Mutations included in the CCR5 coding region may also result ininactivating mutations. In addition, a mutation in the regulatory regionof CCR5 gene may prevent or inhibit expression of CCR5, therebyproviding resistance to some degree from HIV infection.

Once an individual having a homozygous or heterozygous mutant in CCR5 isidentified, it is envisioned that cells from that individual, oncematched for histocompatibility, can be transplanted to an HIV positiveindividual, or to an “at risk” individual.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are to be consideredillustrative and thus are not limiting of the remainder of thedisclosure in any way whatsoever.

EXAMPLE 1 Cloning and Sequence Analysis of the CCR5 Gene

Complementary DNAs were obtained from a λgt11 cDNA library (Combadiereet al., DNA Cell Biol. cit op.). One of the cDNAs obtained, designatedclone 63-2, had a novel sequence highly related to CC CKR2B, but itextended only from bp 105 to 813 of the CC CKR2B open reading frame. The63-2 cDNA was used as a hybridization probe to screen under lowstringency conditions (final wash in 5×SSPE: described by Maniatis etal., Molecular Cloning a Laboratory Manual, Cold Spring HarborLaboratory, at 55° C. for 30 min) a λpCEV9 cDNA library prepared fromendotoxin-stimulated human peripheral blood monocytes as describedpreviously (Combadiere et al., J. Biol. Chem. 270: 29671-5 and J. Biol.Chem. 270: 164914 (1995)). One of the isolated clones, designated clone8.5, matched and extended the sequence of clone 63-2. A 1.4 kb fragmentof the clone 8.5 cDNA was excised from the vector DNA by Bam HI and BstXI double digestion, blunt-ended with Pfu DNA polymerase, subcloned intothe Eco RV site of pBluescript II KS (Stratagene, La Jolla, Calif.), andsequenced completely on both strands. The cDNA insert was then subclonedbetween the Bam HI and Hind III sites of the mammalian expression vectorpREP9 (Invitrogen, San Diego, Calif.).

The clone 8.5 cDNA is 1.4 kb in length. The 5′- and 3′-untranslatedregions and open reading frame are 26,288 and 1056 bp, respectively. The3′-untranslated region is not polyadenylated and lacks a polyadenylationconsensus sequence. The ATG codon proposed to initiate translation isflanked by sequence that conforms favorably with established consensusrules. M. Kozak, Nucleic Acids Res. 15: 8125-48 (1987). The open readingframe contains 352 codons.

Seven segments of the deduced amino acid sequence from SEQ ID NO: 1 havea high content of hydrophobic amino acids consistent withmembrane-spanning domains as well as multiple amino acids conserved inanalogous positions of the known seven-transmembrane-domain receptorrhodopsin. These considerations clearly indicate that CCR5 isancestrally related to rhodopsin-like receptors, and strongly suggestthat it functions as a seven-transmembrane-domain G protein-coupledreceptor. A database search revealed that the highest sequence identityoccurs with chemokine receptors. In particular, the amino acid sequenceof CCR5 is 57, 70, 75, 51 and 48% identical to CC CKR1, CC CKR2A, CCCKR2B, CC CKR3 and CC CKR4, respectively, with lower identity(approximately 30%) to the CXC chemokine receptors, IL-8 receptors A andB. An alignment of the amino acid sequence of CCR5 [“SEQ ID NO: 2”] withthose of CC CKR1 and CC CKR2B is shown in FIG. 1.

The CC CKR2B sequence is eight amino acids longer than CCR5 due to asubstantially longer N-terminal segment. The two sequences are otherwisecolinear with the exception of a four amino acid gap in the putativesecond extracellular loop of CC CKR2B relative to that of CCR5.

Residues of CCR5 that differ from CC CKR2B in the alignment are foundmostly in the putative extracellular domains (the N terminal segment andthe sequence between putative transmembrane domains 2 and 3, 4 and 5,and 6 and 7) and adjacent portions of the transmembrane domains, and inthe C-terminal segment which is predicted to lie in the cytoplasm (FIG.2). Like other seven-transmembrane-domain receptors, the C-terminal tailhas a high content of serine and threonine residues that may be sitesfor receptor phosphorylation as they are in rhodopsin and the β2adrenergic receptor. CCR5 also contains cysteine residues that byanalogy with other seven-transmembrane domain receptors could be a sitefor palmitoylation, tethering this domain to the plasma membrane.

The net charge of the N-terminal extracellular segment of CCR5 is −1.The corresponding domain of CC CKR2B has a net charge of zero, whereasfor other known chemokine receptors this domain is highly acidic. Likeall other known chemokine receptors, CCR5 has conserved cysteineresidues in the N-terminal segment and the third predicted extracellularloop that could form a disulfide bond. Cysteine residues are lessfrequently found in this location in other seven-transmembrane-domainreceptors. CCR5 lacks a consensus sequence for N-linked glycosylation,whereas one or more are found in other CC CKR's. The CCR5 Gene. Whentotal human genomic DNA was digested with Pst I, Eco RI, Hind III andXba I and hybridized with a CCR5 CDNA probe extending from bp 69 to 789relative to the start of the open reading frame, 2-4 bands thathybridized with different intensity were detected in each lane. Theprobe used contains a recognition site for Pst I at bp 532 of the CCR5open reading frame but not for the other restriction enzymes used. Thepattern is most consistent with multiple small cross-hybridizing genes.In fact, restriction fragments of genomic clones isolated for CC CKR2,CC CKR3 and CCR5 can account for all of the bands seen. The CC CKR2B, CCCKR3 and CCR5 open reading frames lack intervening sequences.

EXAMPLE 2 Chemokine Binding to CCR5

Human embryonic kidney (HEK) 293 cells (a total of 10⁷) grown to logphase in DMEM and 10% fetal bovine serum were electroporated with 20 μgof plasmid DNA, and G418-resistant colonies were picked and expanded asdescribed previously (Combadiere et al., op cit.). The cell linesstudied contained large amounts of the recombinant CCR5 mRNA, but lackedCC CKR3 mRNA and native 8.5 mRNA, as assessed by Northern blot analysisof total RNA using full-length cDNA probes. The methods used to createHEK 293 cell lines stably expressing CC CKR1 and CC CKR2B have beendescribed previously (Combadiere et al., J. Biol Chem. 270: 29671-5).

Transfected HEK 293 cells (a total of 10⁶) were incubated in duplicatewith 0.2 nM ¹²⁵I-labeled RANTES, MCP-1, MIP-1α, MIP-1β or MCP-3(specific activity -2200 Ci/mmol, Du Pont/NEN, Boston, Mass.) andvarying concentrations of unlabeled recombinant human chemokines(Peprotech, Rocky Hill, N.J.) in 200 μl of binding medium (RPMI 1640with 1 mg/ml BSA and 25 mM HEPES, pH 7.4). After incubation for 1 h at4° C. or 37° C., unbound chemokines were separated from cells bypelleting through a 10% sucrose/PBS cushion, and the cell-associatedcounts were determined. Specific binding was determined by thedifference in counts in the presence and absence of 1250-fold molarexcess of unlabeled chemokine.

Receptor activation from chemokine binding was assessed by real timemeasurement of [Ca²⁺]_(i) changes using 2 million transfected HEK 293cells loaded with FURA-2. Ratio fluorescence of cells was measured asdescribed previously. Combadiere et al., J. Biol. Chem. 270: 16491-4(1995). J. Van Damme (Rega Institute, Leuven) provided chemicallysynthesized human MCP-2 protein according to a procedure described inProost et al., Cytokine 7: 97-104 (1995). O. Yoshie (Shionogi Institute,Osaka) provided recombinant human eotaxin. Where indicated, cells loadedwith FURA-2 were incubated in holotoxin of B. pertussis (List, Campbell,Calif.) 250 ng/ml for 2 h at 37° C., then washed twice in PBS andresuspended in HBSS. Cell viability was ˜80% by trypan blue exclusionafter pertussis toxin treatment. ATP was purchased from Sigma Co. (St.Louis, Mo.).

Agonists for CCR5. Given the high sequence similarity of CCR5 with otherCC chemokine receptors, the inventors predicted that CCR5 would bespecific for CC chemokines. To test this, the inventors transfected HEK293 cells, which normally are unresponsive to stimulation withchemokines, with the clone 8.5 cDNA and measured the calcium fluxresponses induced by a panel of chemokines. The calcium flux response isstrongly associated with chemotaxis, degranulation and other higherorder leukocyte responses to chemokines, and is a sensitive and specificmeasure of receptor activation. Neither untransfected normock-transfected and selected HEK 293 cells responded to any of thechemokines tested. In contrast, six independent HEK 293 cell linesstably transfected with the clone 8.5 CDNA, three each from two separatetransfections, exhibited [Ca²⁺]_(i) transients in response to MIP-1α,RANTES and MIP-1β, but not in response to MCP-1, MCP-2, MCP-3, eotaxin,IL-8 or γIP-10 all tested at 100 nM. MIP-1α, MIP-1β and RANTES weresimilar in potency and efficacy, the concentrations for half-maximal andmaximal responses ranging from 5-40 and 25-50 nM, respectively. Theseresults indicate that CCR5 is a CC chemokine receptor selective forMIP-1α, MIP-1β, and RANTES.

Desensitization of CCR5. After activation, chemokine receptors havealtered sensitivity to repeated stimulation with the activating agonistand other agonists. When the same chemokine was added in succession toCCR5 transfectants, cells responded to the first addition but not thesecond, indicating that the receptor underwent homologousdesensitization to all three of its agonists. When different agonistswere added in succession, MIP-1α or RANTES given first blocked theresponse to MIP-1β given second, and MIP-1β or RANTES given firstblocked the response to MIP-1α a given second. But MIP-1α or MIP-1βgiven first reduced, but did not eliminate, the response to RANTES givensecond. MCP-1 had no effect on the responses to MIP-1α, MIP-1β orRANTES. These data show a functional interaction of MIP-1α, MIP-1β andRANTES with the same receptor, CCR5.

G Protein Coupling to CCR5. Known chemokine receptors couple toG_(i)-type G proteins, which unlike other classes of G proteins arefunctionally sensitive to pertussis toxin. Treatment of CCR5transfectants with pertussis toxin completely abolished the calcium fluxresponse to MIP-1α, MIP-1β and RANTES. In contrast, the calcium fluxresponse to AT? was largely unaffected. These data indicate that CCR5 inHEK 293 cells is coupled to G proteins of the G_(i) class.

Binding of CC Chemokines to CCR5. The calcium flux results show thatcells expressing CCR5 have acquired the capacity to respond to thepresence of MIP-1α, MIP-1β and RANTES. The mechanism of this effectappears to be related to specific binding to CCR5 on the cell surfacesas judged by radioligand binding assays with intact HEK 293 cells thatwere stably transfected with CCR5 and by comparison with HEK 293 cellsstably transfected with CC CKR1 and CC CKR2B as positive and negativecontrols, respectively. The results for CCR5 are quite complex, and theresults for the positive and negative controls are described first.

The total amounts of ¹²⁵I-MIP-1α, ¹²⁵I-MIP-1β and ¹²⁵I-RANTES that boundto CC CKR2B were similar in magnitude to untransfected HEK 293 cells,and were completely non-specific in both cases, whether the assays werecarried out at 4° C. or 37° C. In contrast, CC CKR2B transfectantsspecifically bound both ¹²⁵I-MCP-1 and ¹²⁵I-MCP-3 at both 4° C. and 37°C. These results are consistent with the known agonists for CC CKR2B.

In the case of CC CKR1, specific binding of ¹²⁵I-MIP-1α was 5-10-foldgreater than non-specific binding at both 4° C. and 37° C., whereasspecific binding of ¹²⁵I-MCP-1 was not detectable. The K_(i) forhomologous competition binding of ¹²⁵-MIP-1α at 4° C. was 10 nM. Theseresults are similar to previously published results, are consistent withMIP-1α's agonist activity for CC CKR1, and represent a positive controlfor MIP-1α binding. Each of the six CCR5 transfectants were tested at 4°C. The total binding of ¹²⁵I-MIP-1α, ¹²⁵I-MIP-1β and ¹²⁵I-RANTES wasequal to the background levels established for the CC CKR2Btransfectant, and was completely non-specific even when radioligandconcentrations as high as 5 nM were tested. Yet, all six cell linesexhibited clear and robust calcium flux responses to MIP-1α, MIP-1β andRANTES.

The G418 concentration in the media of the CCR5 transfectant thatexhibited the strongest calcium flux response was increased from 1 to 3mg/ml for one week. A cell line was derived, named CCR5.1 that exhibitedcalcium flux responses to MIP-1α, MIP-1β and RANTES that wereconsistently double those of the parental cell line. This cell lineexhibited specific binding at 4° C. for MIP-1α, MEP-1β and RANTES.

To increase the sensitivity of the binding assay, non-equilibriumconditions at 37° C. were used to increase the ratio of specific tonon-specific binding for ¹²⁵I-MIP-1α, ¹²⁵I-MIP-1β and ¹²⁵I-RANTES by afactor of 2-4 for both CC CKR1 and CCR5.1 cell lines, compared toresults obtained at 4° C. ¹²⁵I-MCP-1 did not bind specifically to eitherCC CKR1 or CCR5.1 cells at 37° C., whereas ¹²⁵I-MCP-3 bound specificallyto CC CKR1 but not to CCR5.1 cells.

The ¹²⁵I-MIP-1α and ¹²⁵I-MIP-1β binding sites on CC CKR1 and CCR5.1cells were easily distinguished in two ways. First, ¹²⁵-MIP-1α and¹²⁵I-MIP-1β binding to CC CKR1, but not to CCR5.1, was competedeffectively by unlabeled MCP-3. Second, unlabeled MIP-1α competed20-fold more effectively for ¹²⁵I-MIP-1α binding to CC CKR1 than toCCR5.1 (half-maximal inhibitory concentrations IC₅₀ −5 and 100 nM,respectively), and unlabeled MIP-1β competed -2-fold more effectivelyfor ¹²⁵I-MIP-β binding to CCR5.1 than to CC CKR1 (IC₅₀s-100 and 200 nM,respectively).

At 37° C., ¹²⁵I-RANTES bound to both CC CKR1 and CCR5.1 cells at low butsignificantly increased levels compared to the negative control CC CKR2Bcells. Excess unlabeled RANTES reduced ¹²⁵I-RANTES binding to CCR5.1cells (IC₅₀-80 nM). The¹²⁵ I-RANTES binding sites on CC CKR1 and CCR5.1could be distinguished by heterologous competition with excess unlabeledMIP-1α (IC₅₀-20 and 100 nM for CC CKR1 and CCR5.1 respectively).

Distribution of CCR5 RNA. Compared to other CC CKRs, CCR5 is most likeCC CKR2A and CC CKR2B not only in its primary sequence but also in itsRNA distribution. Full-length CC CKR2B and CCR5 open reading frameprobes recognized a 3.5 kb RNA band by Northern blot hybridization intotal RNA made from adherent monocytes. Neither probe recognized RNA inneutrophil or eosinophil samples. To determine whether the CCR5 probewas merely cross-hybridizing to the monocyte CC CKR2 MnRNA, a 30-merantisense oligonucleotide specific for CCR5 was designed. This probealso detected the 3.5 kb monocyte mRNA species. A similar analysis usingspecific oligonucleotide probes has indicated that both CC CKR2A and CCCKR2B RNA is present in adherent monocytes.

EXAMPLE 3 Cell Fusion Assay Suitable for Drug Screening

A vaccinia cell fusion system is used to assay the functional ability ofCCR5 to confer env-mediated fusion competence to CD4-positive nonhumancells. This assay is carried out as described in Nussbaum et al., 1994,supra. In the assay murine NIH 3T3 cells or human HeLa cells are firsttransfected with the plasmid pSC59.CCR5 and then co-infected withvarious vaccinia viruses: vTF7-3 (containing the T7 RNA polymerasegene); vCB3 (containing the human CD4 gene); and vaccinia WR (a negativecontrol). A different cell population is co-infected with variousvaccinia viruses: vCB-21R (containing the E. coli lacZ gene under thetranscriptional control of a T7 promoter (P_(T7)-lacZ)) along witheither a vaccinia virus that encodes a Ba-L env gene from amacrophage-tropic isolate or vCB-16- (a negative control, containing amutant env gene encoding an uncleavable, nonfusogenic unc/env).

The cell populations described above are incubated overnight at 31° C.to allow expression of the vaccinia-encoded proteins. The cells arewashed and mixtures of each combination are prepared in 96-wellmicrotiter plates. Each well contains equal numbers of T7 RNApolymerase-containing cells and lacZ gene-containing cells. Replicateplates are incubated for 24 hours at 37° C. to allow fusion. Samples onone plate are treated with NP-40 and aliquots are assayed forβ-galactosidase activity using a 96-well absorbance reader.

The β-galactosidase assay results from this experiment will show thatNIH 3T3 cells coexpressing human CD4 and CCR5 are highly competent forfusion with cells expressing env from the macrophage-tropic isolate(Ba-L) but not from a T-cell line-tropic isolate (LAV). In contrast, thedata will indicate that NIH 3T3 cells coexpressing human CD4 alone orCCR5 alone are incompetent for fusion with cells expressing env.Furthermore, the low background levels of P-galactosidase produced willindicate that NIH 3T3 cells coexpressing human CD4 and CCR5 do not fusewith cells expressing mutant unc/env.

In a related experiment, several colonies of stable, transformed minkcells that coexpress human CD4 and CCR5 would be tested forsusceptibility to HIV-1 infection by macrophage-tropic or dual-tropicHIV-1 strains (e.g., strains that use CCR5). Transformants containingthe human CD4 gene and an irrelevant control gene are used as negativecontrols. Direct measurements of p24 (HIV core antigen) production willindicate that HIV-l infection is productive with cells that coexpresshuman CD4 and CCR5, but not with the negative controls. Moreover, theefficiency of HIV-1 infection of transformed, CD4-positive,CCR5-positive, nonhuman cells is high enough to be detected directly.

EXAMPLE 4 Anti-CCR5 Antibody Blocks Env-Mediated Membrane Fusion

Based on the known topology of 7-transmembrane segment proteins, fourregions of CCR5 are predicted to be exposed at the cell surface. Naturalor synthetic peptides are produced or synthesized by methods well-knownin the art that correspond to each of these 4 regions. Rabbit antiserais raised by immunization with peptide-KLH (keyhole limpet hemocyanin)conjugates. Total immunoglobulin is purified from the preimmune and theimmune sera by chromatographic separation with Protein-A Sepharose.Alternatively, whole cells expressing CCR5 can be used to generateanti-CCR5 antibodies.

Antibodies raised against an 28 amino acid N-terminal portion of CCR5 oragainst the extracellular loops (e.g., el-1), can block membrane fusionbetween macrophage-tropic strains Ba-L, SF162, JR-FL and ADA of HIV andhuman macrophages, in other words strains that use CCR5. (See, forexample, Feng et al., 1996, supra; Endres et al., 1996, supra).

EXAMPLE 5 Specificity of CCR5 for Env From Macrophage-Tropic Isolates

The sensitivity of fusion mediated by env from different HIV isolates istested with antibodies prepared against the N-terminal portion of CCR5.The anti-CCR5 antibodies inhibit fusion mediated by the prototypicmacrophage-tropic Ba-L env, but will not inhibit fusion mediated by theprototypic T-cell line-tropic LAV env. The fusion inhibition withanti-CCR5 antibodies is not due to nonspecific inhibitory effects on thecells. Coexpression of CCR5 enhances fusion more with env frommacrophage-tropic strains (Ba-L, SF162, JR-FL, and ADA) than with envfrom T-cell line-tropic isolates (IIIB, LAV, and RF).

EXAMPLE 6 CCR5 Peptides Block Env-Mediated Membrane Fusion

Synthetic peptides that correspond to the predicted extracellular loopsof CCR5 were prepared and tested for inhibition of env-mediated membranefusion. Peptides were as follows: extracellular loop-1: LAAQWDFGNTMC(SEQ ID NO:4) extracellular loop-2: RSQKEGLHYTCSSHFPYSQYQFWK (SEQ IDNO:5) extracellular loop-3: QEFFGLNNCSSSNRLD (SEQ ID NO:6)

The peptides were tested using vaccina-based expression and reportergene assay system (see Example 3 above). Cell fusion was quantitated bydetermining the level of β-galactosidase in detergent cell lysates.

FIG. 2 shows that each peptide (0-50 μg/ml) was able to inhibit fusionbetween cells expressing the HIV-1 Env from the macrophage-tropic Ba-Lisolate and murine cells co-expressing CD4 and CCR5.

EXAMPLE 7 Cell Lines Expressing CCR5

Human HeLa, human embryonic kidney (HEK) 293, and murine NIH 3T3 celllines (American Type Culture Collection, Rockville, Md.) were culturedin DMEM-10 (Dulbecco's modified Eagle's medium [Quality Biologicals,Gaithersburg, Md.] containing 10% fetal bovine serum [FBS, HyClone,Logan, UT], 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/nlstreptomycin). The human PM1 T cell line (Lusso et al., 1995) wasobtained from the NIH AIDS Research and Reference Reagent Program(Rockville, Md.) and was grown in RPMI-10 (RPMI 1640 medium [QualityBiologicals] containing 10% FBS, 10 mM HEPES, 2 mM glutamine, andantibiotics). Recombinant vaccinia virus stocks were prepared bystandard procedures (Earl et al., 1991). Pertussis toxin was obtainedfrom List (Campbell, Calif.). Recombinant chemokines were purchased fromPeprotech (Rocky Hill, N.J.). Fura 2-AM and propidium iodide wereobtained from Molecular Probes (Eugene, Oreg.). Sodium azide and ATPwere from Sigma (St. Louis, Mo.).

CCR5 Constructs. Epitope-tagged variants of CCR5 were created to enabledetection by the M5 monoclonal antibody (Kodak, Rochester, N.Y.). TheCCR5 open reading frame was amplified by PCR using the followingprimers: 1) for full-length CCR5 (designated CCR5): a 3′-oligonucleotidecontaining (from 3′ to 5′) 27 bases complementary to the last 9 codonsof CCR5, 3 bases for the stop codon, 6 bases for an Xho I restrictionsite and 8 miscellaneous bases; 2) for CCR5 lacking most of thecytoplasmic C-terminus (designated CCR5₃₀₆): a 3′-oligonucleotidecontaining (from 3′ to 5′) 27 bases complementary to codons 298-306 ofCCR5, 3 bases for a stop codon, 6 bases for an Xho I restriction siteand 8 miscellaneous bases; and 3) for both constructs: a5′-oligonucleotide containing (from 5′ to 3′) 8 miscellaneous bases, 6bases for a Hind III site, 3 bases for the start codon, 24 basesencoding the flag epitope DYKDDDDK and 27 bases complementary to CCR5codons 2 to 10. The resulting two PCR products were digested andsubcloned between the Hind III and Xho I sites of the changes using aMSII fluorimeter (Photon Technology International, S. Brunswick, N.J.)in HEK 293 cell lines expressing receptor constructs as previouslydescribed. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B.(1986). Briefly, cells were loaded with 2 μM FURA-2 AM at 37° C. for 45min, washed twice and resuspended at 10⁶ cells/ml in HBSS, pH 7.4. Twoml of the cell suspension were placed in a stirred, water-jacketedcuvette at 37° C. and excited sequentially at 340 and 380 nm.Fluorescence emission was monitored at 510 nm before and after additionof agonists. For some experiments, cells were incubated with 250 ng/mlpertussis toxin for 3 h prior to functional assay.

Cell Fusion Assay. Fusion between effector cells expressing HV-1 Env andtarget cells expressing CD4 was quantitated by a vaccinia-based reportergene assay in which β-galactosidase is produced selectively in fusedcells (Nussbaum et al., 1994). As effector cells, HeLa cells werecoinfected withn vCB-21R, which encodes the E. Coli LacZ gene undercontrol of the bacteriophage T7 promoter (J. Virol. 70, 5487-5494.), anda recombinant vaccinia virus encoding one of the following HIV-1 Envs(PNAS. 92, 9004-9008.): M-tropic Envs Ba-L (vCB-43; note this is acorrection of the nomenclature used for this virus in Broder and Berger,1995), ADA (vCB-39), SF-162 (vCB-32), and JR-FL (vCB-28); and Unc, anuncleavable mutant of IIIB (vCB-16). In one protocol, the target cellswere HEK 293 cell transfectants stably expressing the indicated CCR5contructs. These cells were coinfected with vTF7-3 encoding T7 RNpolymerase (Fuerst et al., 1993) and vCB-3 encoding human CD4 (Cell 85,1149-12158.). In both viruses the foreign genes are linked to vacciniaearly/late promoters; the multiplicity of infection was 10 pfu/cell foreach virus. In another protocol, the target cells were NIH 3T3 cellstransfected with pcDNA3 -based plasmids encoding CCR5 or CCR5₃₀₆ usingDOTAP lipofection (Boehringer Mannheim, Indianapolis, Ind.); controlcells were transfected with pcDNA3 vector alone. After 4 h incubation inDOTAP at 37° C., cells were coinfected with vTF7-3 and vCB-3; expressionof the CCR5 constructs was driven by the T7 promoter. Cell cultures wereincubated at 31° C. overnight.

Cell surface expression of CCR5 and CCR5₃₀₆ was analyzed by flowcytometry using as the probe either a rabbit polyclonal antiserumgenerated against a synthetic peptide representing the predictedextracellular amino terminal domain of CCR5 (amino acids 1-28), or a mabrecognizing the Flag epitope. Specific cell surface staining atcomparable intensity was obtained when HEK293 cells stably transfectedwith either CCR5 or CCP5₃₀₆ were incubated with the anti-CCR5 antiserum.In contrast, cells stably transfected with the closely related receptorCCR2b (75% amino acid identity) gave only background fluorescenceequivalent to that observed with the signaling is required for the HIV-1coreceptor activity of CCR5, using a quantative vaccinia-based reportergene assay of HIV-1 Env-mediated cell fusion HEK 293 cell transfectantsexpressing CCR5 or CCR5₃₀₆ (along with vaccinia-encoded CD4) were testedfor their ability to fuse with HeLa cells expressing vaccinia-encodedEnvs from several M-tropic strains. Comparable levels of fusion occurredwith CCR5 and CCR5₃₀₆ for each Env tested. Similar results were obtainedin an alternative protocol whereby CCR5 and CCR5₃₀₆ were expressed onNIH 3T3 cells using a transient vaccinia expression system. Thus, theC-terminal truncation that abolished the G protein signal transductionactivity of CCR5 had no effect on fusion coreceptor activity.

Pertussis toxin provided an alternative means to test the requirementfor G protein signal transduction in the fusion coreceptor activity ofCCR5. In the cell fusion assay using the M-tropic Ba-L Env, highconcentration of the toxin (500 ng/ml) had no effect on the fusioncoreceptor activity of CCR5 expressed transiently in NIH 3T3 cells.

The effects of pertussis toxin were also tested on productive HIV-1infection, using the Jurkat-derived T cell line PM1 as the target. PM1cells express CD4 and are highly susceptible to M-tropic HIV-1 strains.Moreover, CCR5 mRNA is expressed in these cells and infection byM-tropic HIV-1 on PM1 cells is CCR5. In the continuous presence ofpertussis toxin (500 ng/ml), robust infection by the M-tropic Ba-Lisolate in PM1 cells was observed. Consistent with this, when PM1 cellswere used as target cells in the cell fusion assay with effector HeLacells expressing the Ba-L Env, high levels of fusion activity wereobserved and this was completely resistant to pertussis toxin. Thus,pertussis toxin at concentrations that potently block G protein-mediatedsignal transduction had minimal effect on either Env-mediated cellfusion or productive infection. These results parallel earlier reportsthat pertussis toxin did not process known as receptor sequestration ordownmodulation. This process is thought to explain in part thephenomenon of receptor desensitization and could be important either forHIV-1 Env-dependent membrane fusion, and/or chemokine inhibition offusion. CCR5 was strongly downmodulated by cheomkine. ligands, whereasthe truncated CCR5₃₀₆ receptor was unaffected. This indicates that, inaddition to containing critical determinants of signaling, theC-terminal domain of CCR5 also contains critical determinants forchemokine-mediated down-modulation.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compositions andprocesses of this invention. Thus, it is intended that the presentinvention cover such modifications and variations, provided they comewithin the scope of the appended claims and their equivalents.

1. An isolated nucleic acid molecule comprising a polynucleotideselected from the group consisting of: (a) a polynucleotide encodingamino acids 1 to 352 of SEQ ID NO: 4; (b) a polynucleotide encodingamino acids 2 to 352 of SEQ ID NO: 4; (c) a polynucleotide encoding apolypeptide having the amino acid sequence encoded by the cDNA containedin ATCC Deposit No. PTA-2425; (d) the complement of (a); (e) thecomplement of (b); and (f) the complement of (c).
 2. The isolatednucleic acid molecule of claim 2, wherein said polynucleotide is (a). 3.The isolated nucleic acid molecule of claim 2, wherein saidpolynucleotide is (b).
 4. The isolated nucleic acid molecule of claim 2,wherein said polynucleotide is (c).
 5. The isolated nucleic acidmolecule of claim 1, wherein said polynucleotide is (d).
 6. The isolatednucleic acid molecule of claim 1, wherein said polynucleotide is (e). 7.The isolated nucleic acid molecule of claim 1, wherein saidpolynucleotide is (f).
 8. The isolated nucleic acid molecule of claim 1,which comprises nucleotides 27-1082 of SEQ ID NO:
 3. 9. The isolatednucleic acid molecule of claim 1, which comprises nucleotides 30-1082 ofSEQ ID NO:
 3. 10. The isolated nucleic acid molecule of claim 1, whichis DNA.
 11. The isolated nucleic acid molecule of claim 1, which is RNA.12. The isolated nucleic acid molecule of claim 1, wherein saidpolynucleotide is fused to a heterologous polynucleotide.
 13. Theisolated nucleic acid molecule of claim 12, wherein said heterologouspolynucleotide encodes a heterologous polypeptide.
 14. A recombinantvector comprising the nucleic acid molecule of claim
 1. 15. Agenetically engineered host cell that comprises the nucleic acidmolecule of claim
 1. 16. A genetically engineered host cell thatcomprises the nucleic acid molecule of claim 1 operatively associatedwith a regulatory sequence that controls gene expression.
 17. Arecombinant method for producing a CCR5 polypeptide, comprisingculturing the recombinant host cell of claim 19 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 18.A recombinant method for producing a CCR5 polypeptide, comprisingculturing the recombinant host cell of claim 20 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 19.A genetically engineered host cell that comprises a nucleic acidmolecule selected from the group consisting of: (a) a polynucleotideencoding amino acids 1 to 352 of SEQ ID NO: 4; (b) a polynucleotideencoding amino acids 2 to 352 of SEQ ID NO: 4; (c) a polynucleotideencoding a polypeptide having the amino acid sequence encoded by thecDNA contained in ATCC Deposit No. PTA-2425.
 20. The geneticallyengineered host cell of claim 19, wherein the nucleic acid isoperatively associated with a regulatory sequence that controls geneexpression.